The present invention relates to a method of selecting a protein variant having reduced immunogenicity as compared with the parent protein, to the protein variant and use thereof, as well as to a method for producing the protein variant.
An increasing number of proteins, including enzymes, are being produced industrially, for use in various industries, housekeeping and medicine. Being proteins they are likely to stimulate an immunological response in man and animals, including an allergic response.
In the present context the terms allergic response, allergy, allergenic and allergenicity are used according to their usual definitions, i.e. to describe the reaction due to immune responses wherein the antibody most often is IgE, less often IgG4 and diseases due to this immune response. Allergic diseases include urticaria, hay-fever, asthma, and atopic dermatitis. They may even evolve into an anaphylactic shock.
Prevention of allergy in susceptible individuals is therefore a research area of great importance. Depending on the application, individuals get sensitized to the respective allergens by inhalation, direct contact with skin and eyes, or injection. The general mechanism behind an allergic response is divided in a sensitization phase and a symptomatic phase. The sensitization phase involves a first exposure of an individual to an allergen. This event activates specific T- and B-lymphocytes, and leads to the production of allergen specific immunoglobulin E (IgE) antibodies. These IgE antibodies eventually facilitate allergen capturing and presentation to T-lymphocytes at the onset of the symptomatic phase. This phase is initiated by a second exposure to the same or a resembling antigen. The specific IgE antibodies bind to the specific IgE receptors on mast cells and basophils, among others, and capture at the same time the allergen. The polyclonal nature of this process results in bridging and clustering of the IgE receptors, and subsequently in the activation of mast cells and basophils. This activation triggers the release of various chemical mediators involved in the early as well as late phase reactions of the symptomatic phase of allergy.
In the present context the antibodies are denoted as usual, i.e. immunoglobulin E is IgE etc.
Various attempts to reduce the immunogenicity of polypeptides and proteins have been conducted. It has been found that small changes in an epitope may affect the binding to an antibody. This may result in a reduced importance of such an epitope, maybe converting it from a high affinity to a low affinity epitope, or maybe even resulting in epitope loss, i.e. the epitope cannot sufficiently bind an antibody to elicit an immunogenic response.
In WO 92/10755 a method for modifying proteins to obtain less immunogenic variants is described. Randomly constructed protein variants, revealing a reduced binding of antibodies to the parent enzyme as compared to the parent enzyme itself, are selected for the measurement in animal models in terms of allergenicity. Finally, it is assessed whether reduction in immunogenicity is due to true elimination of an epitope or a reduction in affinity for antibodies. A drawback of this approach is its xe2x80x98trial and errorxe2x80x99 character, which makes it a long and expensive process. Furthermore, no information is provided on the epitope as such, which implies that the information obtained for one protein cannot be applied on another, since the limited number of animals involved do not allow the identification of epitope patterns.
The present invention relates to a method of selecting a protein variant having reduced immunogenicity as compared with a parent protein, comprising the steps of:
screening a random peptide display package library with antibodies raised against any protein of interest;
sequencing the amino acid sequence of the antibody binding peptides, or the DNA sequence encoding the antibody binding peptides;
identifying epitope patterns by sequence alignment of the reactive peptide sequence;
localization of epitope patterns on the primary and 3-dimensional structure of the parent protein;
defining an epitope area including amino acids situated within 5 xc3x85 from the epitope amino acids;
changing the localized epitope patterns, or amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein without impairing functionality of the protein using the emerging epitope database for eliminating amino acid substitutions creating new or duplicating existing epitope patterns;
introducing the mutated DNA sequence into a suitable host, culturing the host and expressing the protein variant; and
evaluating the immunogenicity of the protein variant using the parent protein as reference.
A second aspect of the present invention is a protein variant having reduced immunogenicity as compared with the parent protein. The amino acid sequence of the protein variant differs from the amino acid sequence of the parent protein with respect to at least one epitope pattern of the parent protein, such that the immunogenicity of the protein variant is reduced as compared with the immunogenicity of the parent protein. Preferably, the immunogenicity of the protein variant of a detergent enzyme is at least 10 times lower than the immunogenicity of the parent protein, preferably 25 times lower than the immunogenicity of the parent protein. In particular, for a protein variant used in personal care products the immunogenicity is preferably 100 times lower, more preferably 200 times lower, even more preferably 500 times lower than the immunogenicity of the parent protein. Furthermore, for food products and pharmaceuticals the immunogenicity is preferably 1000 times lower than the immunogenicity of the parent protein. Normally, a reduction of at most 50,000 times of the immunogenicity is sufficient for all purposes, of protein application. In this context the immunogenicity is assessed by the potential of the protein to evoke an antibody response with focus in IgE responses.
Reduction of immunogenicity in the context above is defined by the differences observed among the kinetics obtained with various doses of the parent protein, as compared with the variants under investigation. In the present study the effect of epitope guided protein engineering is assessed by kinetic studies using a single dose of the protein under investigation.
The immunogenicity of the protein variant is measured in animal tests, wherein the animals are immunized with the protein variant and the immune response is measured. By the present invention the immunogenicity is reduced at least 100 times as compared with the immunogenicity of the parent protein, preferably 200 times reduced, more preferably 500 times.
However, the present inventors have demonstrated that the performance in a competitive ELISA correlates closely to the immunogenic responses measured in animal tests.
To obtain a useful reduction of the immunogenicity or a protein, the immunogenicity of the protein variant must be reduced to at least below 75%, preferably below 50% of the immunogenicity of the parent protein as measured by the performance in competitive ELISA, given the value for immunogenicity of the parent protein is set to 100%. When the immunogenicity is below 50% of the immunogenicity of the parent protein, the protein variant is practically non-allergenic, that is, no IgE response will be measurable in animal tests.
A further aspect of the present invention is a composition comprising a protein variant as defined above, as well as the use of the composition for industrial application, such as the production of a formulation for personal care products (for example shampoo; soap; skin, hand and face lotions; skin, hand and face cremes; hair dyes; toothpaste), food (for example in the baking industry), detergents and for the production of pharmaceuticals.
Yet another aspect is a DNA molecule encoding a protein variant as defined above.
Further aspects are a vector comprising a DNA molecule as described above as well a host cell comprising said DNA molecule.
Another aspect is a method of producing a protein variant having reduced immunogenicity as compared with the parent protein as defined above.